PROJECT SUMMARY Tumor-associated macrophages (TAMs) play an important role in the immune response to cancer, but the mechanisms that control TAMs and T-cell immunity are not completely understood. Glioblastoma (GBM) is the most common primary brain tumor in adults, with a median survival of ~15 months despite aggressive treatment. TAMs constitute more than 30% of infiltrating cells in GBM. Consequently, targeting immune checkpoints in TAMs is considered a promising immunotherapeutic approach for GBM and other tumors. Our data indicate that kynurenine (Kyn) produced by glioma cells controls TAMs and T-cell immunity through the ligand-activated transcription factor aryl hydrocarbon receptor (AHR). Our data demonstrate that: 1) the expression of AHR and AHR-driven genes is upregulated in TAMs in GBM patients and experimental models, and is significantly linked to patient survival; 2) AHR deletion in TAMs significantly decreases tumor growth in experimental GBM; 3) AHR activation by Kyn induces the expression of the transcription factor Krppel-like factor 4 (KLF4) and controls TAM function; 4) AHR also drives the expression of CD39 in TAMs, an ectonucleotidase that promotes the generation of the immunosuppressive metabolite adenosine; 5) CD39 deletion in TAMs ameliorates tumor infiltrating T-lymphocyte (TIL) dysfunction in GBM; 6) A new brain- penetrant AHR antagonist and candidate immune checkpoint inhibitor suppresses growth of several tumors including GBM. Based on these findings, we view AHR in TAMs as a master regulator that responds to oncometabolites (e.g., Kyn) to suppress GBM-specific immunity. Therefore, we hypothesize that AHR in TAMs limits tumor-specific immunity and is a potential immunotherapeutic target for GBM. Our specific aims are:! SPECIFIC AIM 1: Define the role of AHR in the transcriptional control of TAMs. We propose to: 1) Determine if AHR controls TAM polarization via modulation of KLF4 and NF-kB signaling, and 2) Define the transcriptional programs controlled by AHR in TAMs using whole population and single cell approaches. SPECIFIC AIM 2: Study the control of TILs by AHR-driven CD39 expression in TAMs. We propose to: 1) Define the effects of AHR-induced CD39 in TAMs on GBM-specific T cells, 2) Determine if the AHR/CD39 axis controls TILs via adenosine generation, and 2) Dissect the relative contribution of AHR and CD39 in microglia- and peripheral macrophage-derived TAMs to GBM pathology. SPECIFIC AIM 3: Evaluate the therapeutic value of targeting AHR in a GBM preclinical model. We propose to: 1) Study the effects of AHR inhibition on GBM TAMs, 2) Analyze the effects of AHR inhibition on tumor-specific regulatory and effector T cells, and 3) Evaluate the effects of an AHR inhibitor on the immune system in the absence of a direct effect on glioma tumor cells. IN SUMMARY, this project uses unique experimental systems to study a novel pathway that regulates TAMs and T cells in GBM and is a potential therapeutic target for this aggressive and currently incurable disease.